DNA-modified AuNPs Research Articles

Branched DNA-Based Electrochemical Biosensor for Sensitive Nucleic Acids Analysis with Gold Nanoparticles as Amplifier.

A branched DNA-based electrochemical biosensor was designed to sensitively detect specific nucleic acids. On this platform, novel a branched DNA with three sticky ends could be used as a biosensor to sensitively and specifically detect nucleic acids. Meanwhile, we also employed branched DNA-modified AuNPs as a signal amplifier to further improve the sensitivity. Branched DNA sensors, target DNA, and DNA-modified AuNPs formed a sandwich structure to produce an electronic signal for target DNA detection. The reaction primarily involved DNA hybridization without bulky thermal cyclers and enzymes. We proved that the hybridization reaction easily occurred under different conditions, such as the NaCl concentration, reaction time, pH, and temperature, except for a pH lower than 4. The limit of detection could go as low as 0.09 pM (S/N = 3) with excellent specificity and selectivity. There was a correlation curve relationship between the peak current and the logarithm of the target DNA concentration (0.10 pM to 10 nM). The correlation coefficient reached 0.987. The electrochemical platform enables a branched DNA nanostructure to determine nucleic acids for disease diagnosis.

Visual detection of Fusarium temperatum by using CRISPR-Cas12a empowered LAMP assay coupled with AuNPs-based colorimetric reaction

Fusarium temperatum (F. temperatum) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10−8 ng/μL of extracted DNA from F. temperatum. The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.

DNA-functionalized gold nanoparticles: Modification, characterization, and biomedical applications.

With the development of technologies based on gold nanoparticles (AuNPs), bare AuNPs cannot meet the increasing requirements of biomedical applications. Modifications with different functional ligands are usually needed. DNA is not only the main genetic material, but also a good biological material, which has excellent biocompatibility, facile design, and accurate identification. DNA is a perfect ligand candidate for AuNPs, which can make up for the shortcoming of bare AuNPs. DNA-modified AuNPs (DNA-AuNPs) have exciting features and bright prospects in many fields, which have been intensively investigated in the past decade. In this review, we summarize the various approaches for the immobilization of DNA strands on the surface of AuNPs. Representative studies for biomedical applications based on DNA-AuNPs are also discussed. Finally, we present the challenges and future directions.

Rapid colorimetric detection of phthalates using DNA-modified gold nanoparticles

Phthalates, also called phthalic acid esters (PAEs), are well-known endocrine-disrupting chemicals (EDCs) that have been detected in residential and occupational environments. In the present study, a simple and rapid method is proposed for the detection of phthalates (diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP)) based on DNA-modified gold nanoparticles (AuNPs). In the presence of phthalates, the DNA-modified AuNPs formed aggregates. Thus, a visible color change from red to blue and an increase in the absorption ratio with an increasing concentration of phthalates were observed. The limits of detection (LODs) for DEP, DBP, and DEHP were determined to be 0.026 ppm, 0.077 ppm, and 0.144 ppm, respectively. The sensitivity and selectivity test results showed that the DNA-modified AuNPs were able to detect phthalates. The proposed DNA-modified AuNPs (aptamer-E, aptamer-B and aptamer-H) are expected to have potential applications in environmental fields.

DNA-Programmed Bimodal 2D Assembly of Differently Sized Nanoparticles via Folding of Precursory Circular Chains.

Gold nanoparticle (AuNP) assemblies in two-dimensions (2D) exhibit collective physical/chemical properties that are useful for various devices. However, technical issues still impede the efficient ordering of differently sized AuNPs on solid supports while avoiding phase separation. This paper describes a method to construct binary 2D assemblies by folding precursory circular chains composed of small and large AuNPs. The structural change is caused by a spontaneous, non-cross-linking assembly of fully matched double-stranded DNA-modified AuNPs (dsDNA-AuNPs) at a high ionic strength. Since larger dsDNA-AuNPs have a lower critical coagulation concentration of the supporting electrolyte, the spontaneous assembly of large AuNPs precedes that of small AuNPs in the precursory chain during evaporation. Transmission electron microscopy reveals that alternate-type AuNP chains are folded into a binary 2D structure in a mixed mode, whereas block-type chains are transformed into a binary 2D structure in a core-shell mode. The methodology could potentially be harnessed for the fabrication of binary AuNP arrays for various devices.

An enzyme-free electrochemical sandwich DNA assay based on the use of hybridization chain reaction and gold nanoparticles: application to the determination of the DNA of Helicobacter pylori.

An ultrasensitive enzyme-free electrochemical sandwich DNA biosensor is described for the detection of ssDNA oligonucleotides. A DNA sequence derived from the genom of Helicobacter pylori was selected as a model target DNA. The DNA assay was realized through catching target DNA on capture DNA immobilized gold electrode; then labeling the target DNA with reporter DNA (rpDNA) and initiator DNA (iDNA) co-modified gold nanoparticles (AuNPs). The high density of iDNAs serves as one of the amplification strategies. The iDNA triggers hybridization chain reaction (HCR) between two hairpins. This leads to the formation of a long dsDNA concatamer strand and represents one amplification strategy. The electrochemical probe [Ru(NH3)5L]2+, where L stands for 3-(2-phenanthren-9-ylvinyl)pyridine, intercalated into dsDNA chain. Multiple probe molecules intercalate into one dsDNA chain, serving as one amplification strategy. The electrode was subjected to differential pulse voltammetry for signal acquisition, and the oxidation peak current at -0.28V was recorded. On each AuNP, 240 iDNA and 25 rpDNA molecules were immobilized. Successful execution of HCR at the DNA-modified AuNPs was confirmed by gel electrophoresis and hydrodynamic diameter measurements. Introduction of HCR significantly enhances the DNA detection signal intensity. The assay has two linear ranges of different slopes, one from 0.01 fM to 0.5 fM; and one from 1 fM to 100 fM. The detection limit is as low as 0.68 aM. Single mismatch DNA can be differentiated from the fully complementary DNA. Conceivably, this highly sensitive and selective assay provides a general method for detection of various kinds of DNA. Graphical abstractSchematic representation of the detection and the amplification principles of the electrochemical sandwich DNA assay. Purple curl: Captured DNA; Green curl: Reporter DNA; Orange curl: HCR initiator DNA; Yellow solid-circle: Gold nanoparticle; H1 and H2: Two hairpin DNA; [Ru(NH3)5L]2+: Signal probe.

Dithiothreitol-Regulated Coverage of Oligonucleotide-Modified Gold Nanoparticles To Achieve Optimized Biosensor Performance.

DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

Electrochemiluminescence DNA biosensor based on the use of gold nanoparticle modified graphite-like carbon nitride

The authors describe an electrochemiluminescent (ECL) DNA biosensor that is based on the use of gold nanoparticles (AuNPs) modified with graphite-like carbon nitride nanosheets (g-C3N4 NSs) and carrying a DNA probe. In parallel, nanoparticles prepared from gold-platinum (Au/Pt) alloy and carbon nanotubes (CNTs) were placed on a glassy carbon electrode (GCE). Once the g-C3N4 NHs hybridize with DNA-modified AuNPs, they exhibit strong and stable cathodic ECL activity. The Au/Pt-CNTs were prepared by electrochemical deposition of Au/Pt on the surface of the CNTs in order to warrant good electrical conductivity. On hybridization of immobilized capture probe (S1), target DNA (S2) and labeled signal probe (S3), a sandwich-type DNA complex is formed that produces a stable ECL emission at a typical applied voltage of −1.18 V and in the presence of peroxodisulfate. Under optimized conditions, the method has a response to target DNA that is linearly related to the logarithm of its concentration in the range between 0.04 f. and 50 pM, with a 0.018 f. detection limit.

Chiroplasmonic assemblies of gold nanoparticles as a novel method for sensitive detection of alpha-fetoprotein

Serum alpha-fetoprotein (AFP) is present in enhanced concentrations in patients with liver cancer. Thus, sensitive detection of AFP plays an important role in the early diagnosis and treatment of cancer. In this study, a novel chiroplasmonic assay is presented for the determination of AFP. It is based on (a) the use of DNA-induced chiroplasmonic assemblies of gold nanoparticles (AuNPs) and (b) the high specificity and affinity constants between AFP and its aptamer. First, the AFP aptamer is hybridized with its complementary sequence immobilized on AuNP. As a result, the DNA-modified AuNPs are assembled to form AuNPs dimers which display strong chiroptical activity. If, however, AFP is added, it will bind to the aptamer and this results in a destruction of the aptamer-DNA hybrid and a reduction of the CD signal. Under optimized conditions, the CD intensity drops linearly with the log of the concentration of AFP in the range from 0.02 to 5 ng⋅mL−1. Compared to the established method, this assay does not require bisulfite conversion, PCR amplification or separation. Amounts as low as 11 pg⋅mL−1 (S/N = 3) AFP can be detected. The method was applied to spiked human serum samples and gave recoveries ranging from 92.0% to 106%, and the relative standard derivations ranged from 4.6% to 6.3%. To the best of our knowledge, this is the first chirality based assay for AFP.

Easy Design of Colorimetric Logic Gates Based on Nonnatural Base Pairing and Controlled Assembly of Gold Nanoparticles

Utilizing the principles of metal-ion-mediated base pairs (C-Ag-C and T-Hg-T), the pH-sensitive conformational transition of C-rich DNA strand, and the ligand-exchange process triggered by DL-dithiothreitol (DTT), a system of colorimetric logic gates (YES, AND, INHIBIT, and XOR) can be rationally constructed based on the aggregation of the DNA-modified Au NPs. The proposed logic operation system is simple, which consists of only T-/C-rich DNA-modified Au NPs, and it is unnecessary to exquisitely design and alter the DNA sequence for different multiple molecular logic operations. The nonnatural base pairing combined with unique optical properties of Au NPs promises great potential in multiplexed ion sensing, molecular-scale computers, and other computational logic devices.

Synthesis of Stable Peptide Nucleic Acid‐Modified Gold Nanoparticles and their Assembly onto Gold Surfaces

PNA does it better: Peptide nucleic acid (PNA) oligomers were attached to gold nanoparticles (AuNPs) through a variety of mono- and trithiol linkers. These functionalized particles had sufficient stability for sequence-specific self-assembly onto gold surfaces (see figure) in the absence of ions or surfactants. The nanoparticle surface densities obtained were superior to comparable DNA-modified AuNPs.

General Colorimetric Detection of Proteins and Small Molecules Based on Cyclic Enzymatic Signal Amplification and Hairpin Aptamer Probe

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.

Detection of protein–DNA interaction and regulation using gold nanoparticles

The interaction between protein and DNA is usually regulated by a third species, an effector, which can be either a protein or a small molecule. Convenient methods capable of detecting protein–DNA interaction and its regulation are highly desirable research tools. In the current study, we developed a method to directly “visualize” the interaction between a protein–DNA pair and its effector through the coupling with gold nanoparticles (AuNPs). As a proof-of-concept experiment, we constructed a model system based on the interaction between the lac repressor (protein) and operator (DNA) and its interplay with the lac operon inducer isopropyl β- d-1-thiogalactopyranoside (IPTG, which inhibits the interaction between the lac repressor and operator). We coated AuNPs with the lac operator sequences and mixed them with the lac repressor. Because the lac repressor homotetramer contains two DNA binding modules, it bridged the particles and caused them to aggregate. We demonstrated that the assembly of DNA-modified AuNPs correlated with the presence of the corresponding protein and effector in a concentration-dependent manner. This AuNP-based platform has the potential to be generalized in the creation of reporter and detection systems for other interacting protein–DNA pairs and their effectors.

Enzymatic Cleavage of Nucleic Acids on Gold Nanoparticles: A Generic Platform for Facile Colorimetric Biosensors

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.